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[4]	耿 瑶, 尹志良, 李兴平, 肖东琴, 侯伟光. hsa-miRNA-223-3p调控人骨髓间充质干细胞成骨分化的作用[J]. 中国组织工程研究, 2021, 25(7): 1008-1013.
[5]	邹 刚, 徐 志, 刘子铭, 李豫皖, 杨继滨, 金 瑛, 张 骏, 葛 振, 刘 毅. 人脱细胞羊膜支架促进Scleraxis修饰人羊膜间充质干细胞体外成韧带分化[J]. 中国组织工程研究, 2021, 25(7): 1037-1044.
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Currently, the surgery is the major treatment for denervation-like atrophy in weaker ocular muscle of strabismic patients^[1-4] and degenerated patients, but there are many complications, although people keep trying other treatments, such as botulinum toxin and recin mAb35, and also achieve good results, there are still a lot of problems need to solve^[3-7]. Many scholars have injected exogenous basic fibroblast growth factor (bFGF) into the injury or degenerated skeletal muscle, they found a promotion on muscle regeneration, the bFGF gene transfected into skeletal muscle satellite cells can treat myocardial infarction, skeletal muscle injury, muscle atrophy, urinary incontinence and other diseases, and its possible mechanism has been deeply explored^[8-17]. In the previous study, we have successfully isolated and cultured rat extraocular muscle myoblasts^[18-19], and transfected bFGF gene to the myoblasts of rat extraocular muscles and then expressed, the transfected myoblasts secrete bFGF protein, promote cell growth, and increase their survival ability. But how to regulate the transfected bFGF gene expressed in the myoblasts of rat extraocular muscle? Insufficient expression cannot achieve the goal, while overexpression may bring about a series of problems, only controlling the intensity of its expression will provide new ideas and methods for carrying out non-surgical treatment of strabismus. In order to solve this problem, tetracycline induction and control system was introduced into this experiment, to observe the regulatory effect on the transfected bFGF gene expressed in the myoblasts, and to provide evidence for future testing and clinical application. According to the literature, tetracycline induction and control system requires cell stability^[20-23], although the authors have successfully isolated and cultured the rat extraocular muscle myoblasts, also successfully transfected bFGF gene, these cells will be degraded with passage. The biological characteristics of rat extraocular muscle myoblasts are similar with that of skeletal muscle, so in this experiment, C2C12 mouse skeletal myoblasts which have been specially treated and commercialized, was used to replace rat extraocular muscle myoblasts, this kind of cells are able to stabilize the passage without degradation, thus meeting the experimental requirements. The aims of this experiment are intended to clear the following problems: ① The possibility of establishing the myoblasts cell lines stably expressing bFGF. ② Tetracycline can regulate bFGF gene expression in myoblasts. ③ The secretion of target protein when tetracycline induces bFGF gene expression in myoblasts.

Gene therapy is the most researched and the most promising new therapy, and it is producing or has been produced encouraging results on some difficult diseases. The controllability of transgenic expression is one of the key issues for the practical application of gene therapy. Different from common synthetic drugs or products of protein engineering, gene can persistently exist in vivo and exhibit sustained expression, so it must be regulated to ensure its security and effectiveness^[24-25]. The purpose of gene regulation is to connect regulatory genes with target genes or effectively transfer them into target tissues or organs at the same time, target gene expression can be necessary, accurate, fast and efficient, also has no toxicity or antigenicity^[25]. Tetracycline-regulated gene expression system is the most widely used gene regulation system, by adding or removing tetracycline or its derivatives in culture medium or in vivo, it may induce or inhibit the expression of target genes, like a genetic switch, being convenient and useful. Compared to other gene expression systems, tetracycline- regulated gene expression system has many other advantages, its inducer is a kind of antibiotics clinically applied in recent decades, in pre-clinical and clinical phase studies, it is non-toxic and highly secure. Tetracycline, including doxycycline, can be rapidly metabolism and cleared from the body, so fast expressed in gene, the long-term expression and the rapid decline in transgene expression would be the ideal drugs^[25-27]. Tetracycline-regulated gene expression system components can identify single sequence DNA, doxycycline has no hindering effect on the natural proteins^[28-29], so the potential serious side effects can be greatly reduced by using this system. Tight regulation of specific cells, or use of specific cell type or target carrier system to local region, will greatly reduce the potential risk^[21]. Nevertheless, as these proteins are from the bacteria, they are potentially immunogenic^[30], and further study should deeply confirm whether the immune system can recognize Tet-on system components. Tetracycline repressor systems^[31-38], used in this experiment, including the regulation plasmid pcDNA6/TR and inducible expression vector pcDNA4/myc-his, belonging Tet-on system, it is the most advanced and sophisticated international regulation system and has the following advantages: transcriptional activator- trans-acting factor / tetracycline response element almost have no background activity under the condition of no tetracycline; good regulation and induction; fast response to administrating or removing inducer; stringent transgene regulation; small cell toxicity and immunogenicity. The success of the experiment is to connect target gene bFGF with inducible expression plasmid pcDNA4/TO/myc-His^TMA in T-Rex system, to establish tetracycline-induced bFGF gene expression in C2C12 cells, and to regulate the transfected myoblasts secrete or stop secreting bFGF protein, the results will lead us to further improve the various experiments, including animal experiments, hoping transfect the growth factor gene into the weak muscle of the strabismus patients one day, through tetracycline regulation on the expression of growth factor gene in extraocular muscles and secretion of growth factor, it can induce the secondary development on the weak muscle of strabismus patients, the extraocular muscles return to a new balance and re-correct strabismus.

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